Novel tetrahydroisoquinolines as DHFR and CDK2 inhibitors: synthesis, characterization, anticancer activity and antioxidant properties

In this study, we synthesized new 5,6,7,8-tetrahydroisoquinolines and 6,7,8,9-tetrahydrothieno[2,3-c]isoquinolines based on 4-(N,N-dimethylamino)phenyl moiety as expected anticancer and/or antioxidant agents. The structure of all synthesized compounds were confirmed by spectral date (FT-IR, 1H NMR, 13C NMR) and elemental analysis. We evaluated the anticancer activity of these compounds toward two cell lines: A459 cell line (lung cancer cells) and MCF7 cell line (breast cancer cells). All tested compounds showed moderate to strong anti-cancer activity towards the two cell lines. Compound 7e exhibited the most potent cytotoxic activity against A549 cell line (IC50: 0.155 µM) while compound 8d showed the most potent one against MCF7 cell line (IC50: 0.170 µM) in comparison with doxorubicin. In addition, we examined the effect of compounds 7e and 8d regarding the growth of A549 and MCF7 cell lines, employing flow cytometry and Annexin V-FITC apoptotic assay. Our results showed that compound 7e caused cell cycle arrest at the G2/M phase with a 79-fold increase in apoptosis of A459 cell line. Moreover, compound 8d caused cell cycle arrest at the S phase with a 69-fold increase in apoptosis of MCF7 cell line. Furthermore, we studied the activity of these compounds as enzyme inhibitors against several enzymes. Our findings by docking and experimental studies that compound 7e is a potent CDK2 inhibitor with IC50 of 0.149 µM, compared to the Roscovitine control drug with IC50 of 0.380 µM. We also found that compound 8d is a significant DHFR inhibitor with an IC50 of 0.199 µM, compared to Methotrexate control drug with IC50 of 0.131 µM. Evaluation of the antioxidant properties of ten compounds was also studied in comparison with Vitamin C. Compounds 1, 3, 6, 7c and 8e have higher antioxidant activity than Vitamin C which mean that these compounds can used as potent antioxidant drugs. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13065-024-01139-w.


Introduction
Nowadays cancer is one of the most dangerous diseases in the world and it has risen to the position of the leading cause of death around the globed due to the inherent resistance of many types of cancer to conventional radiotherapy and chemotherapy [1].So many strategies have been admitted treating cancer patients.One modality is through inhibition of cell cycle regulators enzymes of cancer cells such as inhibition of CDKs [2] and DHFR enzymes [3], epidermal growth factor (EGF) [2], Ras, and Tubulin proteins [4].CDKs (cyclin-dependent kinases) are serine/threonine kinases enzymes that play a crucial role in regulating eukaryotic cell cycle [5], apoptosis, differentiation, and transcription.So, controlling CDKs activity has emerged as a promising therapeutic approach [5,6].CDK2 is one of CDK families which exist as an inactive form [5,6], upon binding to its regulatory partners cyclin A or cyclin E. Which formed a functional heterodimeric complex to control cell cycle progression [7,8].Previous studies found that CDK2 is over-activated in many types of cancer [8].Which makes CDK2 inhibitions is a desirable target for cancer treatment [9,10].CDK2 inhibitors could be classified as ATP-competitive and non-ATP-competitive based on their binding site [11].Roscovitine and Flavopiridol are the most common commercial CDK2 inhibitors drugs where their structure based on heterocyclic moiety [12].
Dihydrofolate reductase enzyme (DHFR) is responsible for reduction of dihydrofolate (DHF) to tetrahydrofolate (THF).THF is essential for DNA synthesis, cell growth, and the production of raw materials for cell proliferation in both normal and cancer cells [13].Therefor inhibitions of DHFR is an important target to prevent cell spreading [14].Moreover DHFR enzyme required to maintain bacterial growth [15,16].Due to its critical role in nucleotide biosynthesis.Hence inhibitors of DHFR have been proven in as effective agents for treating bacterial infections [16].Methotrexate is the most effective commercial drug for DHFR inhibition which contain heterocyclic atoms.In addition it has been approved to be effective in reducing cancer symptoms in children with acute lymphoblastic leukemia [14,15].
In view of the above observations, the current work was designed to synthesize and characterize some new (5,6,7,8-tetrahydroisoquinolin-3-yl)Thio compounds and related 6,7,8,9-tetrahyrothieno[2,3-c]isoquinolines incorporating 4-(N,N-dimethylamino) phenyl moiety to be examined as anticancer agents and antioxidant drugs.Dimethylamino moiety was chosen in this work because of its remarkable antioxidant activities [34] as they associate to the proton donors active groups in the surfaces like amino or methyl groups.These groups can interact by inter molecular reactions on the surface of DPPH to give antioxidant activities through hydrogen atom transfer reaction [35] in comparison with vitamin C drug.In addition to the tetrahydroisoquinolines anticancer [31,32] properties in comparison with doxorubicin control and compounds 7e and 8d were the most potent compounds.Furthermore, the effect of compounds 7e and 8d on induced apoptosis and cell cycle arrest of the cancer cell lines were also included.Moreover, the enzyme inhibitory activities and molecular docking of two selective tetrahydroisoquinolines 7e and 8d were studied.
Instrumentations: Melting points were determined on a Gallan-Kamp apparatus and are uncorrected.The purity of the compounds was ensured by TLC and the spectroscopic analysis.
IR spectra were recorded on a Shimadzu 470 IRspectrophotometer (KBr; ν max in cm −1 ).The 1 H and 13 C NMR spectra were recorded on Varian A5 500 MHz spectrometer using DMSO-d 6 as a solvent and tetramethylsilane (TMS) as an internal reference.Coupling constants (J values) are given in Hertz (Hz).Elemental analyses were performed on a Perkin Elmer 2400 LS Series CHN/O analyzer.
Cell lines: The in vitro human breast cancerous cell line (MCF7), lung cancerous cell lines (A549) and normal cell lines were purchased from Serum and Vaccine formulation in Cairo-Egypt.
Molecular docking: Molecular docking studies were performed in (I Mole Lab for bioinformatics, Cairo, Egypt).

Biological evaluation In vitro cytotoxic activity
In vitro cytotoxic activity of all synthesized compounds against two human breast cell line (MCF7) and lung cell lines (A549) was evaluated according to the MTT method [23-25, 37, 38].Firstly, Growth the cell line medium in 96 well tissue culture plate was injected with 10 5 cells/mL (100 uL/plate well) of the cell line and incubated at 37 °C for 24 h to develop a monolayer sheet then the formed growth medium was poured from 96 well microtiter plates after the confluent sheet of cells.After that preparing the isoquinoline samples stock solutions in DMSO and diluted the concentrations to started from 0.0487, 0.0975, 0.195, 0.391, 0.781, 1.562, 3.125, 6.25, 12.50, 25.00 µM.Secondly, add 0.1 mL of each concentration tetrahydroisoquinoline sample to each plate.The plates were incubated at 37 °C.Thirdly MTT solution (5 mg/mL in PBS) is prepared.Add 20 µL of MTT solution to each well plates.And shaking in 150 rpm for 5 min, to mix the MTT into the media.Then incubate at (37 °C, 5% CO 2 ) for 1-5 h.Finally read the optical density at 560 nm and subtract background at 620 nm.

Cell cycle analysis
The cell cycle arrests of compound 7e against A549 and compound 8d against MCF7 at their IC 50 values were carried out according to Abcam method (code ab139418), (www.abcam.co.jp).Thus, A549 and MCF7 cells were collected and fixed with 75% ice-cold ethanol before being stored at − 20 °C for 1 h after being treated with an IC 50 dose of our compounds 7e, 8d.Then centrifuged the cells and washed twice with ice-cold PBS, and incubated for 20 min at 4 °C.A cell cycle assay was used to assess the cell cycle (Propidium Iodide Flow Cytometry Kit [ab13941].Then perform statistical analysis for the result by the Cell quest software on the cell fractions in sub-G0/G1, S, and G2/M phases [38].

Annexin-V FITC apoptosis assay
The Annexin-V FITC apoptosis assay of compounds 7e against A549 cell line and 8d against MCF7 cell line at their IC 50 values were carried out according to (BioVision) protocol (code k101-25).(www.biovi sion.com).Thus, cell line were treated with the IC 50 concentration of the compounds for 24 h then collected by trypsin, and centrifuged then rinsed with PBS and suspended in 0.5 mL of binding buffer, then dual-stained with Annexin V-FITC (5 μL) and propidium iodide (5 μL) in the dark for 15 min at RT.These stained cells were measured using flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.The results were then analyzed with the Cell quest software [39][40][41].

Molecular docking
Protein preparation The three-dimensional crystal structures of cyclin-dependent kinase 2 (CDK2, PDB ID 1AQ1) and dihydrofolate reductase (DHFR, PDB ID 1BOZ) were obtained from the Protein Data Bank (PDB).The protein structures were prepared using AutoDock-Tools 1.5.6.All water molecules were removed and hydrogen atoms were added.Gasteiger charges were assigned and nonpolar hydrogen were merged.

Ligand preparation
The 3D structures of ligand 1 (7e compound) and ligand 2 (8d compound) were built and energetically minimized using Avogadro 1.2.0 with the MMFF94 force field.Ligand atom types were assigned and rotatable bonds were defined using AutoDockTools.Both ligands were converted to PDBQT format required for docking calculations.
Molecular docking Molecular docking studies were performed in (I Mole Lab for bioinformatics, Cairo, Egypt) by using AutoDock Vina 1.1.2.For each protein target, a docking grid box was generated to cover the active site based on a co-crystalized ligand.The exhaustiveness parameter was set to 8. Docking was performed with the prepared proteins and ligands to generate 9 binding poses per ligand.The best binding poses based on docking score were visually analyzed using Biovia Discovery Studio 2020 for interactions with key active site residues.
Firstly, prepare the master mixture (6 μL of 5 × Kinase assay buffer 1 + 1 µL of ATP (500 µM) + 5 µL of 10 × CDK substrate peptide 1 + 13 μL of distilled water).thenadd 25 μL of master mixture to every well of the 96-well plate.Add 20 ng of Cyclin A2 and 30 ng of different CDK2 mutant protein into the wells as indicated along with 0.155 µM of our synthesized compound 7e.Incubate at 30 °C for 45 min.After the 45-min reaction, add 50 µL of Kinase-Glo Max reagent to each well.After that cover the plate with aluminum foil and incubate the plate for 15 min at RT. Then Measure luminescence after subtracted The value of blank from all readings using the microplate reader.The relative kinase activity of Cyclin A2/wild-type CDK2 group is set as 100%.The data was analysied and plot by Graphpad prism software [42,43].
Firstly, Dilute 2 μL Dihydrofolate Reductase with798 μL DHFR Assay Buffer.Then add 98 μL of diluted Dihydrofolate Reductase into desired well(s) containing the out synthesized 8d compound.Add 40 μL of diluted NADPH to each well containing the test samples.Incubate at room temperature for 10-15 min.Add 60 μL of diluted DHFR substrate to each well containing the test samples vortex briefly and keep on ice.Measure the absorbance immediately at 340 nm.Then calculate the inhibition concentration of 8d compound.The data was analyzed and plot by Graphpad prism software [44,45].

Characterization
The structures of all newly synthesized compounds were confirmed by FT-IR, 1 H NMR and 13 C NMR as well as elemental analyses (cf.Experimental part section and Additional file 1: Figs.S1-S45).

Anticancer activities In vitro cytotoxicity
Our newly synthesized compounds 1, 3-6, 7a-e, and 8a-e were studied for their in vitro cytotoxic activities against two selective cell lines MCF7 and A549 (which our compounds show high activities towards them by using a way to drug predication program) by using the MTT assay method [36,37].In this work, doxorubicin was used as a positive control drug for comparison with the synthesized compounds under the same experimental conditions.Ten concentrations of each compound and doxorubicin ranging from 0.04875 to 25 μM were tested to reach the concentration which could cause death for 50% of the cancer cells (IC50).The cell viability and toxicity percentage are given in supplementary data (Additional file 1: Tables S1-S6), and summarized in Table 1 and Fig. 1.These results indicated that all synthesized compounds possess high cytotoxic activity against the two cell lines under investigation compared with that of doxorubicin, with IC 50 values ranging from 0.117 to 3.800 μM (Table 1).
In more details on structure-activity relationship, we noticed that: (i) the cytotoxic activity of compounds 1 and 3-6 against MCF7 cells obeys the order 6 > 3 > 5 > 1 > 4, whereas that of the same compounds obeys approximately opposite order against A549 cells as 4 > 5 > 1 > 3 > 6; (ii) 4-substituted phenylcarbamoylmethylthio derivatives 7b-d exhibited stronger cytotoxic activity than the parent unsubstituted one 7a against both MCF7 and A549 cell lines; (iii) among the arylcarbamoylmethylthioisoquinolines 7a-e and arylcarbamoyl thienoisoquinolines 8a-e, naphalen-1-yl derivative 7e exhibited the highest cytotoxic activity against A549 cell line and 4-chlorophenyl derivative 8d showed the highest activity against MCF7 cell line, respectively.Moreover the toxicity of these two compounds against normal human fetal lung fibroblast WI-38 cell line were investigated in this study which show that 7e and 8d compounds not toxic and safe for normal lung cell line with IC 50 19.7 µM, 23.3 µM respectively in comparison with Doxorubicin IC 50 11.43µM (Table 2) the test details presented in Additional file 1: Table S6a.
By calculating the selectivity index of these compounds 7e, 8d and Doxorubicin ((SI) = IC 50 of compound in noncancerous cell line (WI-38)\IC 50 of compound in cancer cell (A549)).They show very high selectivity index SI = 127, 45, 52 respectively.therefore these compounds belong of a selected potential anticancer drugs.Cyclization of arylcarbomyl-methylthioisoquinolines 7a and 7c into the corresponding arylcarbomylthienoiso-quinolines  and A549 cell lines (Fig. 1, and Table 1).

Cell cycle analysis in MCF7 and A549 Cells
The high cytotoxic activity of compound 7e against A549 (IC 50 0.155 µM) and compound 8d against the MCF7 cell line (IC 50 0.170 µM) prompted us to further investigate the growth inhibitory mechanism of the target conjugates to study the mechanism of the cell cycle by using flow cytometric analysis [46][47][48].Both   regulation of cell cycle progression and apoptosis induction have been considered significant strategies to control the proliferation of different cancer cells, accordingly, we primarily examined the growth inhibition mechanism of compounds 7e and 8d in relation to cell cycle progression and regulation in A549 and MCF7 cancer cells, respectively (Fig. 2, and Table 3).The impact on cell cycle distribution was assessed by a DNA flow cytometry analysis, through incubation of A549 cells with compound 7e at its IC 50 concentration (IC 50 0.155 µM) and incubation of MCF7 cells with compound 8d at its IC 50 concentration (IC 50 0.170 µM) for 48 h (Fig. 2).From the obtained results, it was found that: (i) A549 cells exposed to compound 7e significantly arrested at the G2/M phase of the cell cycle with an escalation in G2/M phase fraction from 11.60 (in control cells) to 23.61% (in 7e-treated A549 cells) and (ii) MCF7 cells treated with compound 8d had a significant decrease in G0-G1 and G2/M phases than control cells in contrast (iii) S phase was significantly increased in treated cells as an indication of cell cycle arrest; i.e. increased from 29.81 (control) to 37.92 (8d-treated cells).The antiproliferative mechanism of our compounds was explored from the aforementioned obtained result; compounds of type 7e compound arrested the cell cycle at G2/M phase of the cell cycle whereas compounds of type 8d compound arrested the cell cycle at S phase (Fig. 2).

Apoptosis assay in A549 and MCF7 cell lines
To further investigate whether the anti-proliferative activity for compound 7e or 8d is harmonious with the apoptosis induction [47][48][49][50] within A549 or MCF7 cells pointed out by the increased cell population in G2/M phase in 7e-treated A549 cells and S phase in 8d-treated MCF7 cells, respectively, and AnnexinV-FITC/PI dual staining analysis was used for the apoptosis assay (Fig. 3).
The results of the Annexin V-FITC/PI assay suggested that: (i) treatment of A549 cells with compound 7e led to early and late cellular apoptosis, which proved through the significant increase the percentage of the apoptotic cells in both the early apoptotic phase (from 0.36 to 26.85%) and the late apoptotic phase (from 0.18 to 15.61%) that indicates a high increase in total apoptosis when compared to the untreated control (Fig. 3a, b), (ii) compound 8d caused a considerable increase in early and late apoptosis of MCF7 cells than control cells; i.e. the early and late apoptotic population increased from 0.55 to 22.38% and from 0.27 to 26.96%, respectively (Fig. 3c, d), and (iii) treating A549 cells with compound 7e increases the population of necrotic cell from 1.41 (control) to 3.73% keeping the necrosis minimally contributing.Also, the population of necrotic cells increases from 1.89 (control) to 5.04% upon the subjection of the MCF7 cells to compound 8d (Fig. 4).From the above results, an overall 79-fold increase in A549 cellular apoptosis after treatment with compounds 7e and 69-fold increase in MCF7 cellular apoptosis after treatment with 8d compound In comparison to the control.We observed that our targeted substances, 7e and 8d, have the potential to function as a biological mechanism for inhibiting cell growth, thus leading to cytotoxic effects against the MCF7 and A549 cell line (Fig. 4).

Molecular docking
The docking studies revealed that compound 7e had stronger binding affinity (− 10.3 kcal/mol) to CDK2 compared to the standard STU299 (− 11.5 kcal/mol).The interactions analysis showed that 7e formed hydrogen bonds, amid pi-sulfate, alkyl, pi-alkyl, and pi-sigma interactions with key amino acid residues in the CDK2 binding site like GLU 12, VAL 18, LYS 33, and LEU 134 (Table 4, Fig. 5).In contrast, STU299 showed hydrogen bonds, C-H bonds, alkyl, pi-alkyl, and pi-sigma interactions with residues like GLY 13, GLN 131, LEU 134, VAL 18, ILE 10.The additional pi-sulfate and amid interactions of 7e with GLU 12 likely contribute to its better binding over STU299.
Overall, the docking results indicate compounds 7e and 8d bind more strongly to CDK2 and DHFR respectively compared to the standard inhibitors.The additional interactions formed by 7e and 8d with key active site residues likely contribute to their enhanced binding affinity.

Enzyme inhibitory activities
The promising anti-proliferative impact of compounds 7e and 8d, in addition to their cell cycle disruption and proapoptotic effects, proved a further exploration for their possible inhibitory activities against many enzymes such as RET (encodes a receptor tyrosine kinase) and CDK2 (Cyclin-dependent kinase 2) treated with compound 7e, and DHFR (Dihydrofolate reeducates), Eef2 Kinase (Eukaryotic elongation factor 2kinase)and IKB kinase (inhibitory kappa B kinase) treated with compound 8d.Inhibitory activity of compound 7e towards CDK2 Compound 7e showed significant CDK2 cyclin A inhibitory activity in comparison with the reference; Roscovitine Table 6.Due to the nature of isoquinoline moiety [51,52].From the docking study the inhibition mechanism of compound 7e with interaction with CDK2 with hydrogen bonding and other bonds; they may deactivate the binding site in CDK2 and either its partners or substrates resulting in specific inhibition of CDK2.The obtained results in Table 6 and Fig. 7a and for more enzyme inhibition test details presented in supplementary data (Additional file 1: Table S7) showed that the tested compound 7e exhibited significant inhibitory action against CDK2 with IC 50 value 0.149 ± 0.007 in comparison with the control; Roscovitine which showed IC 50 of 0.380 ± 0.008 µM (reference of CDK2 inhibitor).

DHFR inhibitory activity of compound 8d
Our results obtained indicated that compound 8d which contains tetrahydrothieno[2,3-c]isoquinoline [14,53,54] moiety showed high inhibitory activity towards DHFR enzyme in comparison with the reference; methotrexate show Table 7, Fig. 7b and for more enzyme inhibition test details was presented in supplementary data (Additional file 1: Table S8).Thus, compound 8d exhibited good inhibitory activity towards DHFR with IC 50 value 0.199 ± 0.016 in comparison with that Methotrexate (IC 50 of 0.131 ± 0.007).
Other enzyme inhibitory activity Compounds7e and 8d exhibited moderate inhibitory activity towards other enzymes under investigation in comparison with their control for more enzyme inhibition test details show Additional file 1 (Table 8 and Additional file 1: Tables S9-S11).

In vitro antioxidant behavior
Ten newly synthesized compounds were studied as in vitro antioxidants by measuring of their DPPH scavenging activity which is represented as a percentage % [32] .Results are represented by mean ± SD of three replicates.Table 9 showed the percentage of DPPH scavenging activity of the tested compounds in a dose-dependent relationship compared with Vitamin C (ascorbic acid) as a standard.The higher dose concentration of 0.05 μg/mL represents higher antioxidant activity.Compounds 1, 3, 6, 7c and 8e have higher result than Vitamin C itself.Compound 8e show the highest significant result which suggests that this compound can be used as excellent antioxidant drugs.The high antioxidant activity is referred to the presence of C=O, NH 2 , and OH groups like ascorbic acid [55,56] which can be easily oxidized and reduced and can be used as antioxidant drugs.(Fig. 8 and Table 9).

Conclusion
In this paper, We successfully synthesized and characterized of novel two series of substituted methylthiotetrahydroisoquniolines and related tetrahydrothieno[2,3-c]isoquinolines.All synthesized compounds were evaluated for their anticancer activity towards A549 and MCF7 cell lines, and showed promising results.Moreover, the cell cycle arrest and apoptosis induction of the two representative compounds was studied.Compound 7e caused cell cycle arrest of A549 cell line at G2/M phase and compound 8d arrest the cell cycle of MCF7 cell line at S phase.Compounds 7e and 8d compounds caused high increase in the early and late apoptosis and necrosis.Furthermore, compound 7e showed significant inhibition of CDK2 enzyme while compound 8d exhibited significant activity as a DHFR inhibitors.In the future we intend to synthesis new series of tetrahydrothieno[2,3-c]isoquinolines to studied their anticancer activity not only       in vitro but also in vivo and examined the anticancer activity of these compounds in patient samples as potent anticancer drugs.
Raw date of toxicity and viability of compounds 1,3-6 against MCF7.Table S2.Raw date of toxicity and viability of compounds 7a-e against MCF7.Table S3.Raw date of toxicity and viability of compounds 8a-e against MCF7.Table S4.Raw date of toxicity and viability of compounds 1,3-6 against A549.Table S5.Raw date of toxicity and viability of compounds 7a-e against A549.Table S6.Raw date of toxicity and viability of compounds 8a-e against A549.Table S7.CDK2 inhibitor detailed results.Table S8.DHFR inhibitor detailed results.Table S9.Eef2 inhibitor detailed results.Table S10.IKB inhibitor detailed results.Table S11.RET inhibitor detailed results.

8a and 8c resulted
in increasing the anticancer activity towards both MCF7 and A549 cell lines; (v) cyclization of tolylcarbomylmethylthioisoquinolines 7b into the corresponding tolylcarbomylthieno[2,3-c]isoquinolines 8b decreases the anticancer activity towards MCF7 cell line and (vi) cyclization of carbomylmethylthioisoquinolines 7e into the corresponding carbomylthienoisoquinoline 8e decreases the anticancer activity towards both MCF7

Fig. 2
Fig. 2 Cell cycle analysis of A549 and MCF7 cells treated with compounds 7e and 8d

Fig. 4 3 Table 4
Fig.4  Apoptosis/necrosis assessment of A549 and MCF7 cells after treatment with compounds 7e against A549 and 8d against MCF7.Different cell populations were plotted as a percentage of total events.Data are presented as mean ± SD; n = 3

Fig. 5 Table 5
Fig. 5 3D and 2D docking interaction of compound 7e with CDK2 in compered to the slandered STU299

Fig. 6
Fig. 6 3D and 2D docking interaction of compound 8d with DHFR in compered to the slandered PRD400

Table 2
Cytotoxicity (IC 50) of compounds 7e and 8d and Doxorubicin against normal cell line WI

Table 3
Cell cycle analysis of A549 and MCF7 cells treated with compounds 7e and 8d

Table 8
Enzyme inhibitory activity of compounds 7e and 8d